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  • Saccharomyces cerevisiae  (307)
  • Yeast  (261)
  • Springer  (552)
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  • 2005-2009
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  • 1980-1984  (133)
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 9 (1992), S. 229-234 
    ISSN: 1476-5535
    Keywords: Heat shock protein (HSP) ; Yeast ; Saccharomyces ; Viability ; Thermotolerance ; Ethanol tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Heat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.
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  • 2
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    Journal of industrial microbiology and biotechnology 7 (1991), S. 191-195 
    ISSN: 1476-5535
    Keywords: Yeast ; Trehalose ; Osmotolerance ; Viability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A total of 12 yeast strains from various genera were examined for their ability to produce ethanol in the presence of high concentrations of glucose. From these studies, the yeastsTorulaspora delbrueckii andZygosaccharomyces rouxii were observed to the most osmotolerant. These osmotolerant yeast strains were also observed to possess high concentrations of intracellular trehalose. Futhermore, these strains were found to be tolerant to long-term storage at −20°C and to storage at 4°C in beer containing 5% (v/v) ethanol. Cells containing high trehalose levels at the time of freezing or cold storage exhibited the highest cell viabilities. Trehalose concentration was observed to increase during growth on glucose, reaching a maximum after 24–48 h. Increasing the incubation temperature from 21 to 40°C also resulted in an increase in intracellular trehalose content. These results suggest that trehalose plays a role in enhancing yeast survival under environmentally stressful conditions.
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  • 3
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    Journal of industrial microbiology and biotechnology 7 (1991), S. 263-268 
    ISSN: 1476-5535
    Keywords: Yeast ; β-Glucanase ; β-Glucosidase catabolite repression ; Sporulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The activities of three glycosidases, β-glucosidase and β(1,3)- and β(1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of β-glucanases only increased at the end of the fermentation. The β-glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of β-glucanase activity.
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  • 4
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    European journal of nutrition 22 (1983), S. 205-212 
    ISSN: 1436-6215
    Keywords: Schwermetallwirkung ; Malatdehydrogenase ; Glutamatdehydrogenase ; Glycerinaldehyd-3-phosphatdehydrogenase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Description / Table of Contents: Summary The difference between cadmium, zinc, lead, and mercury in regard of their effects on the activity of the enzymes tested is very slight. Concentrations higher than 10−5 M reduce significantly the activity of the enzymes, and concentrations of approximately 10−3 M inhibit it completely. An increase of the activity cannot be detected. The addition of combinations of cadmium, zinc, and lead results in a summing up of the toxic effects, whereas the interaction between mercury and the other three heavy metals shows a cumulative effect, which is appointed nearly completely by the heavy metal more toxic. The findings suggest that under in-vitro conditions there exists a direct interaction between the heavy metals and the enzymes.
    Notes: Zusammenfassung Die vier Schwermetalle Cadmium, Zink, Blei und Quecksilber unterscheiden sich in ihrer Wirkung auf die Aktivität der untersuchten Enzyme nur sehr wenig. Konzentrationen über 10−5 M vermindern die Enzymaktivität signifikant, und Konzentrationen von etwa 10−3 M unterbinden sie völlig. Eine Steigerung der Enzymaktivität läßt sich nicht feststellen. Die Zugabe von Cadmium-, Zink- und Bleikombinationen führt zu einer Addition der toxischen Effekte, während bei der Interaktion zwischen Quecksilber und den anderen drei Schwermetallen die Gesamtwirkung fast ausschließlich durch das stärker hemmende Schwermetall allein bestimmt wird. Die erhaltenen Ergebnisse lassen vermuten, daß es unter Invitro-Bedingungen zu einer direkten Wechselwirkung zwischen den Schwermetallen und den Enzymen kommt.
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  • 5
    ISSN: 1572-8773
    Keywords: catalase ; copper resistance ; pH-dependent growth ; Saccharomyces cerevisiae ; superoxide dismutase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.
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  • 6
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    Monatshefte für Chemie 111 (1980), S. 355-363 
    ISSN: 1434-4475
    Keywords: Cerulenin ; Fatty acid donor ; Inositol deficiency ; Phospholipid biosynthesis ; Triacylglycerols ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Description / Table of Contents: Zusammenfassung Der Umsatz der Lipide vonSaccharomyces carlsbergensis ATCC 9080 wurde nach Vormarkierung mit3H-Ölsäure und14C-Palmitinsäure untersucht. Inositversorgte Zellen zeigen eine Verschiebung der Fettsäuren von den Triacylglycerinen in die Phospholipide, im besonderen in Phosphatidylcholin und Phosphatidylinosit. Eine verstärkte Übertragung der Fettsäuren von Triacylglycerinen auf Phospholipide konnte festgestellt werden, wenn vormarkierte Zellen auf ein Nährmedium, welches Cerulenin enthielt, übertragen wurde. Cerulenin inhibiert die Fettsäuresynthese und ruft in wachsenden Zellen Fettsäuremangel hervor. Inositdefiziente Hefezellen, welche einen erhöhten Triacylglycerinspiegel aufweisen, verwenden diese Triacylglycerine unter Fettsäuremangelbedingungen ebenfalls für die Synthese von Phospholipiden, besonders von Phosphatidylcholin, Phosphatidyläthanolamin und Phosphatidylserin. Da aus früheren Arbeiten bekannt ist, daß inSaccharomyces carlsbergensis praktisch keine β-Oxidation existiert, können die Triacylglyerine in diesem Hefestamm als Speicher für Fettsäuren angesehen werden, welche zur Synthese von Phospholipiden dienen.
    Notes: Abstract The turnover of lipids was studied in the yeast,Saccharomyces carlsbergensis ATCC 9080, after prelabeling of the cells with [3H] oleic acid and [14C] palmitic acid. In inositol supplemented cells, a redistribution of fatty acids from triacylglycerols to phospholipids (mainly phosphatidylcholine and phosphatidylinositol) could be demonstrated. An increased transfer of fatty acids from triacylglycerols to phospholipids was observed when prelabeled cells were transferred to a growth medium containing cerulenin, which inhibits fatty acid synthesis and thus induces fatty acid deficiency in the growing cells. Inositol deficient cells contain increased levels of triacylglycerols, which are equally well utilized for phospholipid (mainly phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine) synthesis under conditions of fatty acid deficiency. The present results together with the previous finding that β-oxidation is practically absent inSaccharomyces carlsbergensis suggest that in this yeast triacylglycerols function as storage of fatty acids which can be mobilized for phospholipid biosynthesis.
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  • 7
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    Cellular and molecular life sciences 40 (1984), S. 1159-1161 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; 5-trifluoromethyl-6-àzauracil ; yeast cell cultures ; cell division ; inhibition of
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Cell division, as studied in asynchronous cultures of yeast cells, is sensitive to 5-trifluoromethyl-6-azauracil (F3CAzU). Under defined conditions (10 mmoles l−1 F3CAzU) this compound blocks immediately and completely the process of cell division. Using synchronized cells, the time-point at which division process of yeast cell can be inhibited by F3CAzU has been determined. The inhibitor effect of this compound is completely reversed by thymine, thymidine and uracil.
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  • 8
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    Cellular and molecular life sciences 48 (1992), S. 172-178 
    ISSN: 1420-9071
    Keywords: Yeast ; protein degradation ; ubiquitin conjugating enzymes ; signals for proteolysis ; stress response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Selective degradation of cellular proteins serves to eliminate abnormal proteins and to mediate the turnover of certain short-lived proteins, many of which have regulatory functions. In eukaryotes a major pathway for selective protein degradation is ATP-dependent and is mediated by the ubiquitin system. This pathway involves substrate recognition by components of a ubiquitin-protein ligase system, covalent attachment of ubiquitin moieties to proteolytic substrates, and subsequent degradation of these conjugates by a multicatalytic protease complex. Recent genetic evidence suggests that the remarkable selectivity of this process is largely controlled at the level of substrate recognition by the ubiquitin ligase system. InSaccharomyces cerevisiae, ubiquitin-conjugating enzymes UBC1, UBC4 and UBC5 have been identified as key components of this highly conserved degradation pathway. Genetic analysis indicates that ubiquitin-dependent proteolysis is essential for cell viability and that UBC4 and UBC5 enzymes are essential components of the eukaryotic stress response.
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  • 9
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    Cellular and molecular life sciences 48 (1992), S. 1162-1164 
    ISSN: 1420-9071
    Keywords: Polygodial ; warburganal ; antifungal activity ; Candida albicans ; Saccharomyces cerevisiae ; Pityrosporum ovale ; enhancing effect ; antioxidants ; vitamin C ; BHA ; anethole
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The antifungal activity of two drimane sesquiterpene dialdehydes, polygodial (1) and warburganal (2), alone and in combination with several other substances, was examined against three fungi,Candida albicans, Saccharomyces cerevisiae andPityrosporum ovale employing a broth dilution method. Anethole significantly synergized the activity of the two sesquiterpenoids againstC. albicans andS. cerevisiae however, it had only an, additive effect againstP. ovale. By contrast, two antioxidants, ascorbic acid (vitamin C) and BHA (butylated hydroxyanisole), noticeably enhanced the activity of the sesquiterpenoids againstP. ovale, but had no, effect againstC. albicans andS. cerevisiae.
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  • 10
    ISSN: 1476-5535
    Keywords: Lignocellulosic waste ; Yeast ; Ethanol production ; Optimization study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Hardwood hemicellulose hydrolysate has been utilized as a substrate for ethanol production. Among the three different yeasts tested, the best performances have been obtained, in decreasing order, usingPachysolen tannophilus, Candida shehatae andPichia stipitis. Several pretreatments of this raw material have been studied to improve ethanol yields; in one such pretreatment a strain ofP. tannophilus produced ethanol with a yield of 0.29 gethanol/gsugars (gP/gS); which is only 15% less than the values observed with synthetic media. Neither aeration nor acetone addition improved the fermentation of this substrate; in fact, only a marked stimulation of biomass growth has been observed at the expense of both ethanol and xylitol production.
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  • 11
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    Journal of industrial microbiology and biotechnology 7 (1991), S. 131-135 
    ISSN: 1476-5535
    Keywords: Saccharomyces cerevisiae ; Jerusalem artichoke ; High-fructose syrup ; Ethanol ; Immobilized yeast cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The results from this study showed that Jerusalem artichoke juice can be used for the production of very enriched fructose syrup by selective conversion of glucose to ethanol in a continuous process using immobilized cells ofSaccharomyces cerevisiae ATCC 36859. The product contained up to 99% of the total carbohydrates as fructose compared to 76% in the feed. Using Jerusalem artichoke juice supplemented with some glucose a product was obtained with 7.5% w/v ethanol which made ethanol recovery economically favourable. It was found that some fructose was consumed in these continuous processes; the glucose/fructose conversion rate ratio was regulated by the glucose concentration in the product stream.
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  • 12
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    Journal of industrial microbiology and biotechnology 7 (1991), S. 181-189 
    ISSN: 1476-5535
    Keywords: Saccharomyces cerevisiae ; Torulaspora delbrueckii ; Aroma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.
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  • 13
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    Journal of industrial microbiology and biotechnology 7 (1991), S. 35-39 
    ISSN: 1476-5535
    Keywords: 2-Deoxy-d-glucose ; Yeast ; Catabolite repression ; Derepression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.
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  • 14
    ISSN: 1432-0983
    Keywords: 2-oxoglutarate dehydrogenase ; Saccharomyces cerevisiae ; rad52-mediated chromosome loss
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Ogd1 mutants of Saccharomyces cerevisiae are deficient in mitochondrial 2-oxoglutarate dehydrogenase activity; they cannot grow on glycerol and produce an increased amount of organic acids during growth on glucose as substrate. Using gamma ray-induced rad52-mediated chromosome loss the ogd1 mutation can be assigned to chromosome IX. Tetrad analysis of crosses between ogd1 and other markers on chromosome IX revealed that the OGD1 gene maps on the left arm of this chromosome 1.9 cM from his5.
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  • 15
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Orotate phosphoribosyl transferase ; Nucleotide sequence-5-phosphoribosyl 1-pyrophosphate (5PRPP)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Orotate phosphoribosyl transferase (OPRTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.
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  • 16
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    Current genetics 17 (1990), S. 269-273 
    ISSN: 1432-0983
    Keywords: Yeast ; Actin ; Intron ; Antisense RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Using Northern blot analysis we have detected an approximately 840 nucleotide-long RNA which is complementary to the 5′ leader sequence and the first ten nucleotides of the coding sequence of the yeast actin (ACT1) messenger RNA. We have determined two transcription start sites for this actin antisense RNA (ASR1), both within the ACT1 intron, at about 80 and 90 nucleotides downstream from the 5′ splice site. Analysis of a cDNA clone showed that this RNA species overlaps the entire trailer sequence and approximately 20 nucleotides of the coding sequence of the nearby yeast YPT1 gene.
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  • 17
    ISSN: 1432-0983
    Keywords: Yeast ; Mutants ; Cytochrome ; Mitochondria ; Oxidative phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This paper reports studies of bioenergetic modifications in a TTR1 single-nuclear mutant, isolated as resistant to triethyltin, an inhibitor of mitochondrial ATPase, and effective in cAMP-dependent protein phosphorylation. This mutant appears to have lost the wildtype cell ability to respond to a decrease of oxygen concentration in the growth medium by a decrease of cytochrome concentration in the cell. ATP synthesis rate in mutant cells in both the prestationary and stationary phase of growth appeared increased in comparison to wild-type cells, as too was respiration rate. A comparative study of mitochondria extracted from wild-type and from TTR1 mutant cells showed an increase in respiration rate, an increase in ATP synthesis rate, and an increase in TPP+ uptake in mutant mitochondria. The specific ATPase activity, as well as its sensitivity to TET, appears to be similar for mitochondria extracted from both strains. It was proposed that the modification of mitochondrial biogenesis in the TTR1 mutant may be due to a response of the cell to an increase in ATP hydrolysis caused by the mutation. It is also possible that the modification in cAMP-dependent protein kinase regulation which appeared to occur in this mutant affects protein(s) involved in mitochondrial biogenesis.
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  • 18
    ISSN: 1432-0983
    Keywords: Yeast ; Chromosome mapping ; Acidic ribosomal proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary DNA probes from the genes encoding the acidic ribosomal proteins L44, L44′ and L45, as well as from reporter genes for chromosomes IV, VII, XII and XV, have been hybridised to Southern blots of Saccharomyces cerevisiae DNA resolved by pulsed field gel electrophoresis. The protein L44′ and protein L45 genes have been found to hybridise to chromosome IV, identified by the CAT1 gene probe, while the protein L44 probe hybridises with a band containing chromosomes VII and XV, identified by the ATPase 1 and HIS3 genes respectively.
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  • 19
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mutants ; Farnesyl diphosphate synthetase ; Ergosterol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two yeast mutant strains auxotrophic for ergosterol and blocked in farnesyl diphosphate synthetase (EC 2.5.1.1) were isolated. Genetic analysis has shown that these mutant strains carry additional mutations in the ergosterol pathway besides erg20-1 and erg20-2 which affect FPP synthetase. The novel feature of these mutants is their ability to excrete prenyl alcohols (farnesol and geraniol). As geraniol is toxic for yeast cells, the above leaky mutations in FPP synthetase have to be associated with others in the sterol pathway, in order to slow down geraniol synthesis.
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  • 20
    ISSN: 1432-0983
    Keywords: Glucose oxidase ; Aspergillus ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We report the cloning of the Aspergillus niger glucose oxidase gene and its use to elevate glucose oxidase productivity in A. niger by increasing the gene dosage. In addition, the gene has been introduced into A. nidulans where it provides the novel capacity to produce glucose oxidase. A plasmid, in which DNA encoding the mature form of glucose oxidase was preceded by a Saccharomyces cerevisiae secretion signal, effected high-level production of extracellular glucose oxidase in this yeast.
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  • 21
    ISSN: 1432-0983
    Keywords: Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; Argininosuccinate lyase ; Sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The complete nucleotide sequence of the ARG7 gene, coding for argininosuccinate lyase (EC 4.3.2.1), in the fission yeast (Schizosaccharomyces pombe) has been determined. It consists of an open reading frame of 461 codons. The deduced protein has a molecular weight of 51 200 Da. The gene is devoid of introns which is confirmed by the fact that it is expressed in Escherichia coli after spontaneous insertion of a bacterial sequence probably bearing a prokaryotic promoter. A perfect “TATA” box is found at-72 and the major transcription initiation site in Saccharomyces cerevisiae is located at-11 as shown by primer extension experiments. Comparison of the S. pombe lyase with related proteins from other organisms reveals an important degree of conservation except in the carboxyterminal part of the polypeptide. Additionally, a deletion removing 66 amino acids of the carboxy terminus yields an enzyme exhibiting some biological activity. A unique 1500 b transcript was found in S. cerevisiae when the intact gene was present, but the deleted version of the gene gave rise to at least three transcripts of 1800, 2800 and 3900 b.
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  • 22
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    Current genetics 19 (1991), S. 329-332 
    ISSN: 1432-0983
    Keywords: Mismatch repair ; Plasmid integration ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A single base pair mismatch (G:T or A:C) in the CYC1 gene of the integrative plasmid pAB218 stimulates up to a five-fold integration into the yeast chromosome. Analysis of chromosomal sites of plasmid integration suggests that the mismatch-stimulated integration is not targeted as would be expected if crossovers, localised in the region of the mismatch, were a necessary step in mismatch repair. Instead, the observed mismatch-stimulated plasmid integration could be due to potentially recombinogenic structures formed during mismatch repair, such as single-stranded gaps or denatured DNA regions extending around the plasmid molecule.
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  • 23
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Pyrimidine salvage pathway ; Semi-dominant mutants ; FUR1 ; Uracil phosphoribosyl transferase ; Regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In Saccharomyces cerevisiae, the protein encoded by the FUR1 gene is absolutely required for the expression of uracil phosphoribosyl transferase activity. The occurrence of semi-dominant mutations for 5-fluorouracil-(5FU)-resistance at this locus led us to clone and sequence the semi-dominant fur 1–5 allele. A single point mutation, resulting in the substitution of arginine 134 for serine, is responsible for this mutant phenotype. The fur 1–5 allele is transcribed and expressed at the same level as the wild-type allele. But, in contrast with the wild-type, the UPR Tase activity of the fur 1–5 mutant strain is stimulated in vitro by UTP and does not, therefore, correspond to a loss of feedback of UPR Tase activity. We found that uracil, as a free base, induces a significative increase in transcription and UPR Tase activity in a wild-type strain as well as in uracil-overproducing mutants which principally explains the high efficiency of the pyrimidine salvage pathway in S. cerevisiae.
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  • 24
    ISSN: 1432-0983
    Keywords: PET genes ; Yeast ; Mitochondria ; ATP synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This study details the characteristics of two temperature-conditional pet mutants of yeast, strains ts1860 and ts379, which at the non-permissive temperature show deficiencies in the formation of three mitochondrially encoded subunits of the ATP synthase complex. By analysis of mitochondrial translation products, and of mitochondrial transcription in temperature shift experiments from the permissive (22°C) to the non-permissive (36°C) temperature, it was concluded that the nuclear mutations in both mutants primarily inhibit synthesis of ATP synthase subunit 9, and that reductions in subunit 8 and 6 synthesis are secondary pleiotropic effects. Following transfer to 36°C, cells of mutant ts379 display a near complete inhibition of subunit 9 synthesis within 1 h, coincident with a marked reduction in the level of the cognate oli1 mRNA. On the other hand, near complete inhibition of subunit 9 synthesis in strain ts1860 occurs after 3 h at 36°C, at which time there is little change in the level of subunit 9 mRNA. In both mutants the mRNA levels for subunits 6 and 8 are not significantly affected at the time of inhibition of subunit 9 synthesis. Provision of an alternative source of subunit 8, translated extra-mitochondrially for import into the organelle, does not overcome the mutant phenotype of either mutant at 36°C, confirming that subunit 8 is not the sole or primary deficiency in each mutant. The mutants indicate that the products of a least two nuclear genes (designated AEP1 and AEP2) are required for the expression of the mitochondrial oli1 gene and the synthesis of subunit 9. The product of the AEP1 gene (defective in mutant ts1860) is required for translation of oli1 mRNA while the AEP2 product (defective in mutant ts379) is essential either for the stability of oli1 mRNA or for the correct processing of precursor transcripts to the mature message.
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  • 25
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    Current genetics 19 (1991), S. 389-393 
    ISSN: 1432-0983
    Keywords: Yeast ; Pichia inositovora ; Linear plasmids ; Killer toxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Pichia inositovora, strain NRRL Y-18709, which contains three linear double-stranded DNA plasmids, pPinl-1, pPinl-2 and pPinl-3, was cured of these plasmids both by growing the strain in the presence of 50 μg/ml bisbenzimide, and by exposure to ultraviolet light. Both cured and uncured strains were tested for growth on a variety of carbon sources. No differences in growth response were detected, indicating no discernible involvement of the linear plasmids in the catabolism of these compounds. Culture supernatants of Pichia inositovora were shown to contain a substance larger than 100 kDa that is toxic to Saccharomyces cerevisiae, strain GS 1688. Toxin activity was optimal in YEPD assay plates containing 50 mM citrate buffer with a pH between 3.4 and 4.2. Culture supernatants from P. inositovora were also weakly active against Cephaloascus albidus, strain NRRL Y-18710, and Citeromyces matritensis, strain NRRL Y-18711. Concentrated supernatants from cured P. inositovora strains did not exhibit these activities, consistent with the hypothesis that this toxic activity is linear plasmid-encoded. Unlike the wellknown Kluyveromyces lactis system or the newly identified P. acaciae system, P. inositovora strains cured of their linear plasmids do not become detectably sensitive to toxin produced by the wild-type strain, suggesting a nonplasmid-encoded immunity function.
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  • 26
    ISSN: 1432-0983
    Keywords: Yeast ; Molecular cloning ; Nitrogen mustard hyper-resistance ; Choline transport
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    Topics: Biology
    Notes: Summary The recessive hnm1 mutant allele is responsible for hyper-resistance to nitrogen mustard in Saccharomyces cerevisiae. Transformation with a single-copy HNM1 wild-type allele of such hyper-resistant mutants will restore wild-type sensitivity to nitrogen mustard. By contrast the presence of multi-copy vectors containing HNM1, in either a hyper-resistant hnm1 mutant or an HNM1 wild-type, will lead to a novel, mustard-sensitive phenotype unrelated to defects in DNA repair genes. Gene disruption of HNM1 revealed that this gene is nonessential for cells prototrophic for choline (CHO1) but lethal for cells with a cho1 genotype. Sensitivity to nitrogen mustard of wild-type HNM1, but not of hnm1 mutants, depends on the choline content of the growth medium, with cells grown in choline-free medium exhibiting the highest sensitivity. Sequencing of a 300 bp DNA fragment of HNM1 revealed the identity of this gene with the CTR locus, which is responsible for choline transport in Saccharomyces cerevisiae.
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  • 27
    ISSN: 1432-0983
    Keywords: Mutagen hyper-resistance ; Yeast ; Base sequence ; Gene disruption
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    Notes: Summary A multi-copy plasmid containing the SNQ3 gene confers hyper-resistance to 4-nitroquinoline-N-oxide (4NQO), Trenimon, MNNG, cycloheximide, and to sulfometuron methyl in yeast transformants. Restriction analysis, subcloning, and DNA sequencing revealed an open reading frame of 1950 bp on the SNQ3-containing insert DNA. Gene disruption and transplacement into chromosomal DNA yielded 4NQO-sensitive null mutants which were also more sensitive than the wild-type to Trenimon, cycloheximide, sulfometuron methyl, and MNNG. Hydropathic analysis showed that the SNQ3-encoded protein is most likely not membrane-bound, while the codon bias index points to low expression of the gene.
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  • 28
    ISSN: 1432-0983
    Keywords: Gene cloning ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-μm circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-μm circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-μm circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per μg in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-μm circle transform LL20 at a reduced frequency (6,000–16,000 colonies per μg) and YF233 at extremely low frequencies (1–5 colonies per μg). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-μm circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-μm circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-μm circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-μm circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-μm circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.
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  • 29
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Intragenic recombination ; Mutant polypeptides
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    Notes: Summary Recombinational analysis of oxil mutants was performed using a and a mutant strains with the same mitochondrial and nuclear backgrounds, derived from strain 777-3A. In spite of minor inconsistencies the overall map of oxi1 mutations can be constructed on the basis of wild-type recombinant frequencies in the two-point oxi1 − − x oxi1 − crosses. The frequencies of wild-type recombinants varied in a wide range from 0.003% to 16%, reaching the maximal values expected for unlinked mitochondria) markers. No distinct clusters of mutants were observed. The analysis of translation products of oxil mutants showed that all but one of the oxil mutants studied are connected with the conspicuous changes of the polypeptide band corresponding to subunit 11 of cytochrome c oxidase in electrophoresis on polyacrylamide gels. The exceptional G565 mutant showed no conspicuous change in subunit II, but lacked subunit I of cytochrome c oxidase. Various oxi1 mutants seemed to carry premature chain termination mutations. Most of them show a correlation between the length of the putative fragment of subunit II synthesized and the position on the genetic map. The direction of translation is from the V2 to the V60 mutation. The V2 mutation is proximal to cap and V60 proximal to the par locus.
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  • 30
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    Current genetics 2 (1980), S. 61-67 
    ISSN: 1432-0983
    Keywords: Axenomycin ; Ribosome genetics ; Yeast ; Protein synthesis
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    Notes: Summary Axenomycin inhibits protein synthesis in vivo and in vitro in Saccharomyces cerevisiae. The antibiotic acts by binding to ribosomes, most probably to the large ribosomal subunit. Mutant strains resistant to axenomycin appear to contain ribosomes that are not inhibited by the antibiotic. The responsible gene has been mapped on the VII chromosome between the centromere and the leu1 gene.
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  • 31
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    Current genetics 18 (1990), S. 401-403 
    ISSN: 1432-0983
    Keywords: Baking yeast ; Saccharomyces cerevisiae ; Dough leavening ; Benomyl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary To investigate the leavening ability of yeast in dough, chromosome loss was induced by benomyl treatment in YOY1037, a diploid between a baking strain and a laboratory strain, and its effect on the leavening ability was studied. When benomyl-treated cells were spread on plates with a dye indicator for ploidy, about 20% of the visible colonies were stained dark blue or dark purple; the rest stained pale blue, similar to the diploid YOY1037. Strains showing the MATα phenotype, and non-galactose fermenting strains, apparently having lost particular chromosomes, were observed only in those with darkcoloured colonies. Strains with dark-coloured colonies showed a wider range of leavening ability than did those with pale-coloured colonies.
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  • 32
    ISSN: 1432-0983
    Keywords: Xylitol dehydrogenase gene ; Pichia stipitis ; Saccharomyces cerevisiae ; Xylose utilization
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    Topics: Biology
    Notes: Summary A P. stipitis cDNA library in λgt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.
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  • 33
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Centromere flanking sequences ; tRNA modification enzymes
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    Notes: Summary Transcriptional analysis of the region flanking the left boundary of the centromere of chromosome VI revealed the presence of a gene immediately adjacent to CEN6. The transcription of the gene is directed toward the centromere, and nucleotide sequence analysis showed that the coding region terminates only 50 bp away from CEN6. Our results extend to chromosome VI the observation that centromere-flanking regions of S. cerevisiae are transcriptionally active. Disruption of the coding region of the gene showed that its product, whilst not essential for cell viability, is important for normal cell growth. The gene has been termed DEG1 (DEpressed Growth rate). Comparison of the deduced amino acid sequence of DEG1 with a protein sequence databank revealed homology with the enzyme tRNA pseudouridine synthase I of E. coli.
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  • 34
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    Current genetics 19 (1991), S. 89-94 
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Intron ; Mobile
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    Topics: Biology
    Notes: Summary The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S. cerevisiae mitochondrial introns (Sc cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K. lactis mitochondrial intron (Kl cox1.2). Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities. This distribution is suggestive of recent sequence transfer between species. The remaining S. cerevisiae introns cross react with an additional species but with no set pattern. Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA. These introns are, therefore, mitochondria-specific. Seven strains of K. lactis exhibit striking variability in intron content. In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K. lactis actin gene and the S. cerevisiae RP29B gene) are not detected beyond their source species.
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  • 35
    ISSN: 1432-0983
    Keywords: Yeast ; Pyruvate decarboxylase ; Gene expression ; Codon usage ; Gene fusion
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    Notes: Summary Three structural genes encode the pyruvate decarboxylase isoenzymes in the yeast Saccharomyces cerevisiae. PDC1 and PDC5 are active during glucose fermentation where PDC1 is expressed about six times more strongly than PDC5. Expression of PDC6 is weak and seems to be induced in ethanol medium. Consequently, pdc1Δ pdc5Δ double mutants do not ferment glucose and do not grow on glucose medium. Spontaneous mutants, derived from such a pdc1 pdc5 strain, were isolated which could again ferment glucose. They showed pyruvate decarboxylase activity due to a duplication of PDC6. The second copy of PDC6 was expressed under the control of the PDC1 promoter, which was still present in the pdc1 strain. However, the resulting PDC1-PDC6 fusion gene could only partially substitute for PDC1: to achieve normal growth and high pyruvate decarboxylase activity strains carrying PDC1-PDC6 required a functional PDC5 gene which is dispensable in a PDC1 wild-type background. Thus, expression of PDC5 depends on the state of the PDC1 locus: low in the PDC1 wild-type background and high in PDC1-PDC6 fusion strains and, as shown previously, in pdc1 mutants. The activation of PDC5 expression in PDC1-PDC6 strains may be due to particular properties of the PDC1-PDC6 fusion protein or simply to the weaker expression of PDC1-PDC6 in comparison to the wild-type PDC1 gene.
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  • 36
    ISSN: 1432-0983
    Keywords: AEP2 ; Yeast ; Mitochondria ; ATP synthase
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    Notes: Summary The temperature-conditional pet mutant, ts379, of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature due to mutation of a single nuclear locus, AEP2. The inability to synthesize subunit 9 correlates with a lowered accumulation of the cognate oli1 mRNA indicating that the AEP2 product is involved in oli1 transcript maturation or stabilization. The AEP2 gene has been isolated in this study from a wild-type yeast genomic library by genetic complementation of ts379 at the restrictive temperature. A 1740 nucleotide open-reading frame was observed that encodes a basic, hydrophilic protein of 67534 Da which possesses a putative mitochondrial address signal. Disruption of chromosomal DNA within this reading frame produced a non-conditional respiratory mutant unable to synthesize subunit 9, identifying the AEP2 gene. Hybridization analyses indicate that AEP2 is located on chromosome XIII and produces a 2.1 kb poly(A)+ transcript. Two additional open-reading frames were found in close proximity to that of AEP2. The three open-reading frames shared no significant homology with entries in several data bases.
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  • 37
    ISSN: 1432-0983
    Keywords: Yeast ; Ribosomal protein gene ; Sequence analysis
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    Notes: Summary Two genes encoding ribosomal protein YL41 were cloned from Saccharomyces cerevisiae chromosomal DNA. Both genes contain an uniterrupted region of only 75 nucleotides coding for a protein of 3.3 kD. Within the coding regions the nucleotide sequences are virtually identical, whereas in both the 5′-and 3′-flanking regions the two genes differ significantly from each other. The deduced protein shows an arginine and lysine content of 68 percent, i.e., 17 out of 25 residues, and the basic residues are evenly distributed over the molecule. When compared to the ribosomal protein sequences currently available no counterpart to YL41 could be found in prokaryotes and it seems likely that YL41 is a eukaryotespecific ribosomal protein.
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  • 38
    ISSN: 1432-0983
    Keywords: Yeast ; Trans-acting Factor ; RAP1
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    Notes: Summary We show by deletion mutagenesis, followed by in vivo and in vitro analysis, that the binding of a protein factor to the upstream activation sequence (USA) of the Saccharomyces cerevisiae glycolytic gene PYK, encoding pyruvate kinase, is required for efficient transcription of the corresponding coding region. In addition, gel electrophoretic mobility shift and DNase I protection studies, involving yeast gene products expressed in E. coli, suggest that this trans-acting DNA-binding protein is encoding by the RAP1 gene. The identification of RAP1 binding sites located within the UAS element of the yeast PYK, PGK (phosphoglycerate kinase) and ENO1 (enolase) genes, and in the 5′-upstream region of the ADHI (alcohol dehydrogenase) gene, suggests that a mechanism of coordinate gene expression involving several of the glycolytic genes may exist in yeast.
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  • 39
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    Current genetics 18 (1990), S. 485-491 
    ISSN: 1432-0983
    Keywords: Yeast ; Ty elements ; Virus like particles
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    Notes: Summary Electron microscopic analysis of thin sections of Saccharomyces cerevisiae NCYC74 has revealed the presence of many clumped cytoplasmic particles that morphologically resemble Ty element virus-like particles (VLPs). Accumulation of Ty VLPs has only previously been observed in S. cerevisiae strains that over-express a cloned Ty element. The particles in NCYC74 co-purify with Ty RNA, Ty-specific antigens and a reverse transcriptase activity. Furthermore, they appear to be recognised by antibodies to Ty VLPs during indirect immunofluorescence experiments. These observations provide compelling evidence that the cytoplasmic particle in NCYC74 are indeed Ty VLPs.
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  • 40
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    Current genetics 18 (1990), S. 29-39 
    ISSN: 1432-0983
    Keywords: Recombination ; DNA repair ; UV irradiation ; Yeast
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    Notes: Summary A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1–3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.
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  • 41
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondrial RNA splicing ; Nuclear pet - mutant ; Group I introns
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    Notes: Summary We have studied the role of the product of the nuclear gene PET157 in mitochondrial pre-mRNA splicing. Cytoduction experiments show that a mitochondrial genome deleted for the three introns bI3, aI5 and aI6 is able to suppress the pet157-1 mutation: the strain recovers respiratory competency indicating that the product of the PET157 gene is only required for mitochondrial premRNA splicing. Characterization of the high molecular weight pre-mRNAs which accumulate in the pet157 mutant demonstrate that the product of the PET157 gene is required for the excision of two group I introns bI3 and aI6 (corresponding to aI5β) located in the cob-box and coxI genes respectively. Furthermore, the pet157 mutant strain accumulates the bI3 maturase in the form of a polypeptide of 50K (p50) previously observed in mitochondrial mutants defective in the excision of bI3. We have shown by restriction analysis and allelism tests that the pet157-1 mutation is allelic to the nuclear mrs1 mutation, previously described as specifically blocking the excision of bI3. Finally, revertants obtained by the deletion of bI3 or aI6 from the mitochondrial DNA were isolated from the MRS1 disrupted allele, confirming the involvment of the product of the MRS1/PET157 gene in the excision of the two introns bI3 and aI6.
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  • 42
    ISSN: 1432-0983
    Keywords: Mutagen hyper-resistance ; Nitrogen mustard ; Saccharomyces cerevisiae
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    Notes: Summary A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementations group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.
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  • 43
    ISSN: 1432-0983
    Keywords: S. cerevisiae ; Yeast ; Cadmium resistance ; CAD2 gene
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    Notes: Summary A cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae. The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2. The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II. The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains. The results suggest that the novel type of cadmium resistance controlled by CAD2 does not involve production of a cadmiumm-metallothionein.
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  • 44
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; G418 resistance ; Gene cartridges ; Heterologous Gene expression
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    Notes: Summary Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
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  • 45
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    Current genetics 2 (1980), S. 115-120 
    ISSN: 1432-0983
    Keywords: Galactose fermentation ; Saccharomyces cerevisiae ; Regulatory mutant
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    Notes: Summary A novel type of regulatory mutation for galactose metabolism in Saccharomyces cerevisiae is described. The mutation named gal11 was recessive, non-allelic to GAL4, GAL80, GAL2, or GAL3, and unlinked to the gene cluster of GAL1, GAL10, and GAL7. It caused a ‘coordinate’ reduction of galactokinase, galactose-1-P uridylyl transferase, and UDP-glucose 4-epimerase by a factor of more than 5, rendering the mutant cells galactose-nonfermenting. The effect of the mutation was manifested not only in cells grown on galactose but also in cells constitutively synthesizing the galactose-metabolizing enzymes.
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  • 46
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    Current genetics 19 (1991), S. 9-14 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mevalonate kinase ; Ergosterol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48 500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.
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  • 47
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    Current genetics 2 (1980), S. 207-210 
    ISSN: 1432-0983
    Keywords: Yeast ; Plasmid ; Repair
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    Notes: Summary We have developed a system for assaying pyrimidine dimers in the 2 ⇐m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m−2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.
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  • 48
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    Current genetics 2 (1980), S. 211-214 
    ISSN: 1432-0983
    Keywords: Yeast ; Ribosomal protein dimorphism
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    Notes: Summary Two-dimensional gel electrophoresis was used in a comparative study of ribosomal proteins from various strains of Saccharomyces cerevisiae. The results demonstrated a case of dimorphism of L8 protein of 60S ribosomal subunit. Of eight strains examined, two strains were one type and six were the other type. The former, which was tentatively designated as altered (A) type, was more acidic than the latter, common (C) type, as shown by mobility difference in pH gradient gel. Heterozygous (A/C) diploid cells contained both types of L8 protein and gave rise to tetrads of 2:2 segregation for A and C types, indicating that the difference of mobility was reflection of the allelic difference of the gene coding for L8 protein.
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  • 49
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    Current genetics 2 (1980), S. 223-228 
    ISSN: 1432-0983
    Keywords: Transcriptional Units ; GAL Genes ; Saccharomyces cerevisiae ; UV mapping
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    Topics: Biology
    Notes: Summary The size of the transcriptional unit of the structural genes for three galactose-metabolizing enzymes which form a cluster on chromosome II in Saccharomyces cerevisiae was studied by the ultraviolet light (UV)-mapping technique. Thus the size of the primary transcripts of GAL7 for galactose-1-phosphate uridylyl transferase, GAL10 for uridine diphosphoglucose 4-epimerase, or GAL1 for galactokinase were estimated to be 0.81 x 106, 1.1 x 106, or 1.3 x 106 respectively. In the light of these data together with the known directions of transcription of the genes, we concluded that each of three genes was transcribed from its own promoter.
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  • 50
    ISSN: 1432-0983
    Keywords: Pulsed field gel-electrophoresis ; S1 nuclease sensitive sites ; Repair ; Yeast
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    Notes: Summary Repair under non-growth conditions of DNA double-strand breaks (DSB) and chromatin sites sensitive to S1 endonuclease (SSS) induced by 60Cobalt-gamma rays were monitored in repair-competent and deficient strains of Saccharomyces cerevisiae by pulsed field gelelectrophoresis. In stationary-phase cells of a repair-competent RAD diploid, and an excision-deficient rad3-2 diploid, SSS are repaired as efficiently as DSB, whereas in a repair-competent RAD haploid, and a rad 50-1 diploid, neither SSS nor DSB are repaired. The rad18-2 diploid repairs DSB well but is defective in SSS repair. Obviously, SSS repair in yeast chromatin, like DSB repair, depends on recombination, but unlike DSB repair depends additionally on RAD18 function.
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  • 51
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    Current genetics 1 (1980), S. 177-183 
    ISSN: 1432-0983
    Keywords: Yeast ; Ribosomal protein alteration ; Cycloheximide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected. Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13.
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  • 52
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Episomal plasmid ; Copy number control ; Plasmid maintenance ; Glycolytic enzyme levels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This study demonstrates how varying the promoter strength of an essential gene on a yeast 2μORI-STB YEp multicopy vector can influence vector copy levels. A phosphoglycerate kinase gene (PGK) on this plasmid was made essential for fermentative growth by transformation into a pgk - yeast strain. When in these PGK- transformants the requirement for PGK expression was the sole selective criterion for plasmid maintenance, PGK promoter activity was inversely related to vector copy levels. Plasmids with an efficiently-transcribed PGK gene were maintained at approximately one copy per cell, whereas those lacking the UAS that normally directs high basal PGK transcription levels were present at up to 10–15 copies. All cultures of these PGK+ transformants contained only a low proportion of pgk - cells. Since mitotic loss of the plasmid arrests growth through loss of a functional PGK allele, PGK confers high stability to the YEp vector in such a pgk - genetic background. In this system YEp vector levels are probably influenced by PGK transcription because high expression of PGK is needed in rapid fermentative growth. Remarkably, low plasmid PGK promoter activity caused PGK mRNA levels slightly higher than those found in yeast with normal PGK regulation. A higher plasmid copy number is therefore not the only factor counteracting the effects of low PGK transcription, and it is possible that PGK mRNA becomes more stable in response to inefficient PGK transcription.
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  • 53
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    Current genetics 17 (1990), S. 179-183 
    ISSN: 1432-0983
    Keywords: Heme ; Cytochromes ; Regulation ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Simultaneous effects of mutations in the transcriptional regulatory genes, HAP1, HAP2 and HAP3, on all respiratory cytochromes of Saccharomyces cerevisiae were determined. Cytochrome behavior in hap mutants and in cyc4 and rhm1 mutants, altered in regulation of 5-aminolevulinate synthase, was compared. Although hap mutants were isolated as trans-acting, transcriptional regulators of the CYC1 (iso-1-cytochrome c) gene, each mutant exhibits partial deficiencies in all cytochrome types. In hap2 and hap3 strains all cytochromes were decreased proportionally to about 40–50% of wild type values. In contrast, hap1 caused a decrease in all cytochromes and an accumulation of a pigment, probably Zn porphyrin. Apparently apocytochrome and heme biosynthesis retain coordination in hap2 and hap3, but not in hap1, mutants. Unlike cyc4 and rhm1 mutants, hap mutants do not exhibit 5-aminolevulinate-dependent restoration of cytochromes. The hap1 mutant grew at nearnormal rates on glycerol, whereas hap2 and hap3 mutants grew very slowly. The frequency of [rho-] was high (16–18%) in hap2 and hap3 strains. Results are consistent with generalized control of mitochondrial replication directed by the HAP1-HAP2 system and heme-directed control of formation of all apocytochromes mediated by HAP1. Neither system exerts all-or-nothing control.
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  • 54
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    Current genetics 17 (1990), S. 275-280 
    ISSN: 1432-0983
    Keywords: Yeast ; DNA replication ; Effect on mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.
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  • 55
    ISSN: 1432-0983
    Keywords: Yarrowia lipolytica ; 7SL RNA ; Essential genes ; Yeast
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    Topics: Biology
    Notes: Summary Cells containing a deletion of either the SCR1 or SCR2 genes, which code for the 7SL RNA component of the signal recognition particle (SRP) homologue, were found to be viable. Two independent approaches demonstrated that cells containing deletions of both genes were inviale. Therefore, Yarrowia lipolytica contains two (and only two) functional 7SL RNA genes.
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  • 56
    ISSN: 1432-0983
    Keywords: Mitotic recombination ; Hyper-recombination ; RAD50 ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.
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  • 57
    ISSN: 1432-0983
    Keywords: Flow cytometry ; Rhodamine 123 ; Respiratory chain ; Yeast
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    Topics: Biology
    Notes: Summary The cationic lipophilic dye Rhodamine 123 (Rh123) is selectively enriched in mitochondria in a membrane potential-dependent manner. Application of drugs which interfere with the electron flow of the respiratory chain lead to a severe reduction of mitochondrial dye uptake. In this communication we show that the same effect is observed after Rh123-staining of respiratory-deficient yeast mutants. Based on this observation we used flow cytometry to discriminate respiratory-compentent and respiratory-deficient yeast cells. Combined with a cell sorter we were able to selectively enrich respiring and non-respiring yeast cells, repectively, from a mixture of cells.
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  • 58
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Sporulation ; Inessential genes ; Genome organization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame. SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores. Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions. SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.
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  • 59
    ISSN: 1432-0983
    Keywords: Yeast ; Mistranslation ; ψ-factor
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    Topics: Biology
    Notes: Summary Chromosomal omnipotent suppressor mutations recovered in ψ+ strains of Saccharomyces cerevisiae were brought into ψ− cytoplasm. SUP46, SUP138 and SUP139 acted as dominant omnipotent suppressors in the ψ− cytoplasm though their suppressor activity was substantially reduced. SUP46 and SUP138 conferred recessive thermosensitivity and antibiotic sensitivity in ψ− cytoplasm as in ψ+ cytoplasm. On the other hand, sup111 through sup115, which acted as recessive omnipotent suppressors in the ψ+ cytoplasm, manifested no, or very low, suppressor activity in the ψ− cytoplasm. They, however, still enhanced the efficiency of the SUP29 tRNA suppressor in ψ− cytoplasm. A multicopy plasmid carrying the wild-type SUP35 gene enhanced the efficiency of sup111 in ψ− cytoplasm.
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  • 60
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nucleo-mitochondrial interactions ; Mitochondrial status ; Lycorine
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    Topics: Biology
    Notes: Summary In a previous paper we have shown that the alkaloid lycorine inhibits growth of rho +, mit - and rho -, strains of Saccharomyces cerevisiae, whereas strains devoid of mitochondrial DNA (rho o) are resistant to more than 200 μg/ml of the alkaloid. In this report we show that hypersuppressive petites are almost as resistant as rho o mutants, whereas isogenic rho - petites, which have retained tained longer segments of the genome, are sensitive to the drug.
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  • 61
    ISSN: 1432-0983
    Keywords: Yeast ; QH2: cytochrome c oxidoreductase ; Mitochondrial biogenesis ; Transcription
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    Topics: Biology
    Notes: Summary In Saccharmmyces cerevisiae, the COR2 gene codes for the 40 kDa subunit II of the QH2: cytochrome c oxidoreductase, an enzyme of the mitochondrial respiratory chain. Regions in the 5′ flank of this gene important for regulated expression were identified by assaying β-galactosidase activities in cells carrying different COR2-lacZ fusion genes. Sequences downstream of position-201 relative to the translational initiation codon are sufficient to confer regulation by carbon source, whereas sequences downstream of position-153 do not give rise to significant expression. A binding site for the abundant general transcription factor GFI is present in the region between-201 and-153 just upstream from sequences which resemble the consensus DNA recognition sequence of the regulatory protein complex HAP2/HAP3: 5′-TNATTGGT-3′. By quantitating RNA levels and assaying β-galactosidase activities we show that synthesis of COR2, which is not a hemoprotein, is regulated by HAP1, HAP2/HAP3 and heme.
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  • 62
    ISSN: 1432-0983
    Keywords: Yeast ; Mutant ; Triethyltin chloride ; Protein phosphorylation
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    Topics: Biology
    Notes: Summary Three mutants of Saccharomyces cerevisiae resistant to triethyltin (an inhibitor of mitochondrial ATPase) on non-fermentative media, and non-resistant to this drug on fermentative media, were isolated and named TTR1, TTR2 and TTR3. Apart from triethyltin resistance, these mutants show the following common characteristics: (1) Increased intracellular cytochrome c concentration. (2) Increased respiration rate. (3) Decreased growth yield. (4) Increased growth sensitivity to several drugs inhibiting oxidative phosphorylation: namely, CCCP (permeabilizing inner mitochondrial membrane to protons), valinomycin (permeabilizing inner mitochondrial membrane to potassium) and oligomycin (inhibitor of mitochondrial ATPase). (5) Increased sensitivity to carbon source starvation. For each mutant, these characteristics appeared to be due to a single pleiotropic nuclear mutation. Mutation TTR1 causes additional phenotypic characteristics which do not appear in mutants TTR2 and TTR3: (1) Pinkish coloration of colonies which is more pronounced after a long growth period. (2) Inability of the cells to store glycogen. (3) Growth defect of the cells on a galactose-containing medium. (4) Inability of a diploid homozygote mutant strain to sporulate. All these phenotypic characteristics have already been described in yeast mutants deregulated in cAMP-dependant protein phosphorylation. Crossing of a strain bearing the TTR1 mutation with a strain mutated in the adenylate cyclase structural gene suggested that the TTR1 phenotype is due to a modification in regulation of cAPK by cAMP, making cell multiplication possible without intracellular cAMP.
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  • 63
    ISSN: 1432-0983
    Keywords: Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; CaMV 35S promoter ; CaMV 35S terminator ; Heterologous expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial β-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator. We show here that S. pombe initiates transcription at exactly the same start site as was reported for tobacco. The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct. Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay. Interestingly, expression of the 35S promoter in the budding yeast S. cerevisiae was found to be only moderate and about hundredfold lower than in S. pombe. To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast. In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S. pombe. Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.
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  • 64
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    Current genetics 17 (1990), S. 493-497 
    ISSN: 1432-0983
    Keywords: Mitochondria ; Yeast ; Petites
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    Topics: Biology
    Notes: Summary A pleiotropic, respiration-deficient mutant was isolated from the petite negative yeast Pachysolen tannophilus after UV mutagenesis. The mutant is unable to utilize xylose, arabinose, galactose or glycerol, and shows no detectable respiration when grown on glucose. Cytochrome c oxidase, xylose reductase and xylitol dehydrogenase activities are lacking. Mitochondrial ultrastructre is altered. The results support the hypothesis that functioning mitochondria are necessary for xylose utilization in this organism.
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  • 65
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondria ; Intron-encoded proteins ; Recombination
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    Topics: Biology
    Notes: Summary The respiratory competency of a yeast strain devoid of mitchondrial introns is quite normal. However, it may be asked whether intron-encoded proteins participate in metabolisms other than those of mitochondrial introns. Using strains without mitochondrial introns we have answered two questions. The first was: does the absence of intron-encoded proteins abolsh mitochondrial recombination? The second was: do mitochondrial introns and intron-encoded proteins play a part in mitochondrial DNA rearrangements induced by ethidium bromide (rho- production)? We have shown that the introns and intron-encoded proteins are not essential essential components of either phenomenon.
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  • 66
    ISSN: 1432-0983
    Keywords: Yeast ; Ty2 ; Protein/DNA binding ; Transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5′ regulatory region of this gene. Various Ty-containing His+ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His- and a weak His+ strain was a single point mutation, an A→G transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this A→G transition as being reponsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this A→G mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His+ (“on”) but not His- (“off”) Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.
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  • 67
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Cytochrome c oxidase ; Post-translational regulation
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    Topics: Biology
    Notes: Summary Biogenesis of functional cytochrome c oxidase in yeast requires the product of the nuclear gene SCO1. Strains deleted for this gene fail to accumulate the mitochondrially-synthesized cytochrome c oxidase subunits I and II, despite the presence of the respective mRNAs. Here we present data which demonstrate that the observed phenotype does not result from a failure to translate the mRNAs, but from a preferential degradation of the newly synthesized subunits. The SCO1 protein is therefore involved in a post-translational step in the accumulation of cytochrome c oxidase subunits I and II. We propose that the SCO1 protein is required for the correct assembly of both subunits into the cytochrome c oxidase complex.
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  • 68
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    Current genetics 18 (1990), S. 23-27 
    ISSN: 1432-0983
    Keywords: Protein translocation ; Saccharomyces cerevisiae ; Peroxisomes ; Overexpression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Import of proteins into organelles usually requires a cis-acting targeting signal. Analysis of various hybrid proteins, consisting of mouse DHFR and parts of catalase A from Saccharomyces cerevisiae, revealed that fusion proteins containing the N-terminal 126 amino acids, or less, of catalase A remain in the cytosol whereas fusion proteins containing 140, or more, N-terminal amino acids of catalase A form large aggregates inside the cell. These protein bodies, which lack a surrounding membrane, copurified with peroxisomes on cell fractionation. The peroxisomal targeting signal of catalase A does not reside at the C-terminus or at the N-terminus.
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  • 69
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    Current genetics 2 (1980), S. 175-180 
    ISSN: 1432-0983
    Keywords: Yeast ; Hydroxyurea ; DNA synthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Newly synthesised DNA molecules the same size as replicons (7 million-60 million daltons) accumulate in yeast cells treated with hydroxyurea. During prolonged incubation in low concentrations of the drug, there is a large accumulation of these molecules without any corresponding increase in their molecular weight. On release from the inhibtion the molecules are converted to large molecular weight DNA. These observations are consistent with an inhibition by hydroxyurea of the joining of completed replicons. In addition, newly synthesised DNA molecules the size of yeast Okazaki fragments also accumulate in cells treated with hydroxyurea.
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  • 70
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    Current genetics 2 (1980), S. 193-200 
    ISSN: 1432-0983
    Keywords: Recombination ; Plasmids ; Transformation ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary [2 μm+ and [2μm°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 μm yeast DNA plasmid in addition to the yeast nuclear LEU2 + gene and the Co1E1 derivative, pMB9. In the [2 μm+] transformants, a new wholly yeast LEU2 + plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 μm DNA. The plamid, pYX, in the absence of 2 μm DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 μm DNA portion of the plasmid. pJDB219 was found to require the presence of 2 μm DNA to undergo this intramolecular recombination. The results suggest that 2, μm DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.
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  • 71
    ISSN: 1432-0983
    Keywords: Yeast ; Mutant ; p-Fluoro-dl-phenylalanine ; β-Phenethyl-alcohol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary p-Fluoro-dl-phenylalanine (PFP)-resistant mutants which produce a large amount of β-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, tyr1-pfp, caused both PFP resistance and β-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA.
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  • 72
    ISSN: 1432-0983
    Keywords: Peptides ; Transport ; Regulation ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The transport of small peptides into the yeast Saccharomyces cerevisiae is subject to complex regulatory control. In an effort to determine the number, and to address the function, of the components involved in peptide transport and its regulation, spontaneous mutants resistant to toxic di- and tripeptides were isolated under inducing conditions. Twenty-four mutant strains were characterized in detail and fell into two phenotypic groups; one group deficient in amino acid-inducible peptide uptake, the other with a pleiotropic phenotype including a loss of peptide transport. Complementation analysis of recessive mutations in 12 of these strains defĩned three groups; ptr1 (nine strains), ptr2 (two strains), and ptr3 (one strain). Isolation and screening of 31 additional N-methyl-N-nitro-N-Nitrosoguanidine (MNNG)-induced, peptide transport-deficient mutants produced one ptr3 and 30 ptr2 strains: no additional complementation groups were detected. Uptake of radiolabeled dileucine was negligible in ptr1 and ptr2 strains and was reduced by 65% and 90% in the two ptr3 mutants, indicating that all strains were defective at the transport step. We conclude that the S. cerevisiae amino acid-inducible peptide transport system recognizes a broad spectrum of peptide substrates and involves at least three components. One gene, PTR3, may play an indirect or regulatory role since mutations in this gene cause a pleiotropic phenotype.
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  • 73
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    Journal of molecular evolution 38 (1994), S. 1-17 
    ISSN: 1432-1432
    Keywords: HSP70 ; Heat shock ; Evolution ; Phylogeny ; Yeast ; Multigene family ; Subcellular compartmentalization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups.
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  • 74
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    Journal of molecular evolution 32 (1991), S. 396-404 
    ISSN: 1432-1432
    Keywords: Yeast ; Mitochondrial DNA ; Polymirphism ; Repeated sequences
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A spontaneously arising mitochondrial DNA (mtDNA) variant ofSaccharomyces cerevisiae has been formed by two exta copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA squence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400–500 bp whereas differences in 1–2 bp occur approximately every 60 bp. Some polymorphisms are associated wit optional G+C-rich sequences (GC clusters). Two such optional GC clusters and one A+T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity inS. cerevisiae mtDNA are discussed.
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  • 75
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    Journal of molecular evolution 32 (1991), S. 439-442 
    ISSN: 1432-1432
    Keywords: Yeast ; Mitochondrial DNA ; ori ; rep ; Polymorphism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Threeori elements (ori 2,ori 5, andori 7) have been sequenced inSaccharomyces cerevisiae strain Dip 2 and compared to the equivalentori elements of a second strain (B). Bothori 2 andori 5 exhibit 98% base matching between strains Dip 2 and B. In contrast, the thirdori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within theori structure in Dip 2. This represents a novel polymorphic form of the yeast mitochondrial genome.
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  • 76
    ISSN: 1432-1432
    Keywords: Thiolase ; Peroxisome evolution ; Bootstrap analysis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes.
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  • 77
    ISSN: 1432-1432
    Keywords: Yeast ; E. coli ; tRNA ; rRNA ; Sequence homologies ; Evolution ; Origins ; Coding mechanism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Many tRNAs ofE. coli and yeast contain stretches whose base sequences are similar to those found in their respective rRNAs. The matches are too frequent and extensive to be attributed to coincidence. They are distributed without discernible pattern along and among the RNAs and between the two species. They occur in loops as well as in stems, among both conserved and non-conserved regions. Their distributions suggest that they reflect common ancestral origins rather than common functions, and that they represent true homologies.
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  • 78
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    Journal of molecular evolution 38 (1994), S. 363-368 
    ISSN: 1432-1432
    Keywords: Saccharomyces cerevisiae ; 2-μm circle ; DNA sequencing ; Horizontal transmission ; Site-specific recombination ; Selfish DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 μm) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-μm variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-μm plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection.
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  • 79
    ISSN: 1573-0832
    Keywords: Nystatin ; amphotericin B ; amphotericin B methyl ester ; polyene antibiotics ; yeast ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K+ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K+ and before compounds of higher molecular weights.
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  • 80
    ISSN: 1572-9699
    Keywords: 2-Deoxy-D-glucose transport ; polyphosphate ; Saccharomyces cerevisiae ; sugar phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The role of polyphosphate in 2-deoxy-D-glucose transport was studied in yeast cells, pulse-labeled with [32P]orthophosphate, by comparing the concentrations and specific activities of polyphosphate, orthophosphate and 2-dGlc-phosphate. When 2-dGlc transport was measured under aerobic conditions, it appeared that polyphosphate replenished the orthophosphate pool, indicating that polyphosphate has, at least mainly, an indirect role in sugar phosphorylation. Also in cells with a reduced respiratory capacity, due to a treatment with antimycin A, no direct role for polyphosphate in 2-dGlc transport could be detected. Under these conditions, only a very limited breakdown of polyphosphate occurred, probably because of the small decrease in the orthophosphate concentration.
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  • 81
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    Antonie van Leeuwenhoek 62 (1992), S. 35-46 
    ISSN: 1572-9699
    Keywords: introns ; pre-mRNA splicing ; RNA processing ; Saccharomyces cerevisiae ; yeast genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The occurrence of introns in nuclear precursor RNAs (pre-mRNAs) is widespread in eukaryotes, and the splicing process that removes them is basically the same in yeasts as it is in higher eukaryotes. Splicing takes place in a very large, multi-component complex, the spliceosome, and biochemical studies have been complicated by the large number of splicing factors involved. This review describes how genetic approaches used to study RNA splicing inSaccharomyces cerevisiae have complemented the biochemical studies and led to rapid advances in the field.
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  • 82
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    Applied microbiology and biotechnology 16 (1982), S. 75-80 
    ISSN: 1432-0614
    Keywords: Immobilization of yeast ; Saccharomyces cerevisiae ; Ethanol production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.
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  • 83
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    Cellular and molecular life sciences 46 (1990), S. 193-200 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; protein toxin ; yeast toxin precursor ; protease processing ; lectin ; (1→6)-β-D-glucan ; receptor ; resistant mutants ; spheroplasts ; ion-permeable channels ; site-directed mutagenesis ; toxin functional domains
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The K1 killer toxin ofSaccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.
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  • 84
    ISSN: 1432-0983
    Keywords: Trans-kingdom conjugation ; DNA integration ; Saccharomyces cerevisiae ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary IncQ-derived conjugative shuttle vectors, which carried the yeast gene URA3 and/or the yeast autonomously replicating sequence (ARS1), were constructed. Both the ars-plus plasmid pAY205 and the ars-less plasmid pAY201 were successfully transmitted from E. coli to S. cerevisiae by the action of mob and tra. In this trans-kingdom conjugation, plasmid pAY205 could replicate and be retained in transconjugants. Plasmid pAY201 caused the formation of “micro-colonies” of abortive transconjugants due to its transient expression and rapid disappearance. Nevertheless, one per about 103 colonies caused by transmitted pAY201 plasmids were uncurable by integration into the homologous region of a yeast chromosome. Analyses by restriction enzyme mapping and Southern hybridization indicate that this integration is primarily caused by a double crossover during conjugation and not by a single reciprocal recombination.
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  • 85
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Cytochrome oxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have analyzed a mutation in the mitochondrial gene oxi3 coding for subunit I of cytochrome-oxidase in the yeast Saccharomyces cerevisiae. This mutation replaces one of the seven invariant histidines of the polypeptide (position 378) by a tyrosine, and leads to a respiratory deficient phenotype. A total of 157 revertants, which have recovered the ability to grow on a respiratory substrate, have been selected from this mutant (tyrosine 378). The nature of the reversion has been analysed by a rapid screening procedure and 32 of the revertants have been sequenced. They are all true backmutations reintroducing the histidine in position 378. This very exceptional situation suggests that this histidine is a ligand of the redox center of cytochrome oxidase.
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  • 86
    ISSN: 1432-0983
    Keywords: Yeast ; DNA-polymerase α ; Cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The 5′ ACGCGT3′ MluI motif, which is found in the upstream region of several yeast DNA-synthesis genes which are periodically expressed during the mitotic cell-cycle, is present twice in the 5′ non-coding region of the DNA-polymerase α gene (POL1). Deletion, of the most distal repeat does not affect POL1 transcription, while the adjacent 40 base-pair (bp) downstream sequence is necessary both for the proper level and the fluctuation of POL1 mRNA. This region contains the 5′ACGCGTCGCGT3′ sequence, which is sufficient to control periodic transcription of a CYC1-lacZ reporter gene with the same kinetics observed for POL1. The adjacent 29 bp AT-rich region does not show any activity by itself, but it acts synergistically in conjunction with at least one MluI hexamer to stimulate CYC1-lacZ expression. By further deletion analysis, DNA sequences necessary to initiate POL1 transcription at the proper sites have also been identified.
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  • 87
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    Current genetics 21 (1992), S. 203-206 
    ISSN: 1432-0983
    Keywords: Yeast ; Galactokinase ; Mutant selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The galactose analogue 2-deoxy-galactose (2DG) has been widely used to select for mutations in the gene encoding the galactose pathway enzyme galactokinase (GalK). We have tested the effect of 2DG on Candida albicans to see if it could be used to obtain GalK- mutants in this diploid asexual yeast. 2DG was shown to be toxic to wild-type cells. Enzyme assays demonstrated that 2DG can induce GalK as efficiently as galactose. Examination of the initital rate of galactose uptake indicated that the galactose transport system is constitutive. 2DG-resistant mutants were isolated from mutagenized cultures and shown to have very low levels of GalK activity. The potential genetic applications of this system of direct mutant selection are discussed.
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  • 88
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Transcriptional activator ; Oxidative stress ; Glutathione
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H2O2-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H2O2 promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.
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  • 89
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondrial trp-tRNA synthetase ; Nuclear mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The conditional respiratory-deficient Saccharomyces cerevisiae mutant pet-ts2281 was complemented by an yeast genomic DNA library. The gene thus isolated was sequenced and proved to be identical to the known MSW1 sequence encoding mitochondrial tryptophanyl-tRNA synthetase (Myers and Tzagoloff 1985). Compared to the wild-type, the ts2281 mutant allele of MSW1 contained a single T→C transition leading to a Leu→Ser replacement at position 294 of the protein sequence. In addition to this mutational alteration, our sequence data for the wild-type gene differ from the originally published MSW1 sequence at five other DNA positions which affect two locally restricted regions of the polypeptide chain. As expected, at the non-permissive temperature ts2281 cells are specifically defective in mitochondrial trp-tRNA formation and, thus, in overall mitochondrial protein synthesis. In addition, the patterns of cytochrome b mRNA maturation intermediates were distinctly different in ts2281 and wild-type yeast cells. The mutational effect of the observed amino-acid substitution in ts2281 is discussed in terms of weakened hydrogen bonding in the C-terminal half of the MSW1-encoded protein.
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  • 90
    ISSN: 1432-0983
    Keywords: Repressor ; Zinc finger ; Leucine zipper ; GATA-1 ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The UGA43 gene of Saccharomyces cerevisiae is required for repression of inducible genes involved in the utilization of 4-aminobutyric acid (GABA) or urea as nitrogen sources. The UGA43 gene has been cloned by complementation of a uga43 mutation. The N-terminal region of the UGA43 protein is very similar to the DNA-binding zinc-finger region typical of the GATA regulatory factor family in vertebrates. UGA43 is the first reported instance of a GATA protein with a negative regulatory function. The C-terminal region of the predicted UGA43 protein contains a putative leucine zipper. Sequencing of three uga43 mutant alleles suggests that the GATA and putative leucine-zipper regions are both required for the repressive activity of UGA43. UGA43 appears to be a highly regulated gene. On “poor” nitrogen sources, UGA43 transcripts are measured at high levels whereas they are nearly undetectable in conditions of nitrogen catabolite repression. The levels measured on “poor” nitrogen sources are further increased in uga43 mutant cells, suggesting that UGA43 exerts negative autoregulation.
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  • 91
    ISSN: 1432-0983
    Keywords: Yeast ; cAMP ; RAS ; GAP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation. Homologous integration of this DNA in the genome occurs at the CCS1 locus. An 11 kb of the DNA insert is necessary for complementation. Sequencing part of the fragment identifies CCS1 as the IRA2 gene. The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins. This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function. We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.
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  • 92
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    Current genetics 24 (1993), S. 21-25 
    ISSN: 1432-0983
    Keywords: Yeast ; Glycerol kinase ; GUT1 ; ADR1 control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The GUT1 gene of Saccharomyces cerevisiae, encoding glycerol kinase, was cloned and sequenced. The cloned genomic DNA fragment contains an open reading frame potentially coding for a protein of 709 amino acids with homology to bacterial glycerol kinases (40.8% identity over 502 amino acids, and 42.1% identity over 496 amino acids, in comparison to the smaller E. coli and B. subtilis enzymes). Disruption of GUT1 showed that the gene is required for growth on glycerol, but not on glucose or ethanol media. No glycerol kinase activity was detected in the disruption mutant. According to enzyme activity and transcript analysis, synthesis of glycerol kinase is repressed by glucose, and derepression is ADR1-dependent.
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  • 93
    ISSN: 1432-0983
    Keywords: Yeast ; FLP ; Phase variation-type expression ; Gene replacement
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of a neomycin phosphotransferase II (NPTII) gene has been designed to be regulated by an FLP-mediated switching of the orientation of the NPTII coding region located on the invertible DNA segment in episomal yeast plasmids. Inversion of the segment from inverted to direct orientation with respect to the promoter resulted in a dramatic increase in G418 resistance. FLP also promoted a double reciprocal exchange between the transforming and the resident 2-μm plasmid, leading to insertion of the FLP and REP2 genes into the transforming plasmid. The results demonstrate a possible use of FLP recombinase for ‘phase variation’-type regulation of gene expression and gene replacement in eukaryotic cells.
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  • 94
    ISSN: 1432-0983
    Keywords: Glucoamylase ; Gene cloning ; Hormoconis resinae ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37–48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.
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  • 95
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    Current genetics 26 (1994), S. 95-99 
    ISSN: 1432-0983
    Keywords: Translational fidelity ; Paromomycin ; Stuttering ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Missense errors in the translation of mRNAs in Saccharomyces cerevisiae were screened by looking for charge heterogeneity of proteins on two-dimensional gels resulting from the substitution of charged and neutral amino acids. No such mistranslation was detected in wild-type yeast strains grown in the presence of the translational error-inducing antibiotic paromomycin. However, paromomycin-induced mistranslation of a heterologous mRNA, encoding human phosphoglycerate kinase expressed in yeast, was seen. We suggest that the combination of error-prone translation of a heterologous mRNA, and growth in the presence of paromomycin, leads to an accumulation of mistranslated proteins that can be detected by two-dimensional gel electrophoresis.
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  • 96
    ISSN: 1432-0983
    Keywords: DNA repair ; Heat shock ; Hyperthermia ; Mutagenesis ; pso3-1 mutant ; Psoralen ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A putative tolerance, induced by heat shock (HS), to the lethal and mutagenic effects of 8-methoxypsoralen (8-MOP) photoaddition and hyperthermia was analyzed in Saccharomyces cerevisiae using the wild-type strain N123 and the isogenic DNA repair-deficient mutant pso3-1. In wild-type cells, the HS (38°C for 1 h) did not modify either the survival or the mutation frequency observed after 8-MOP photoaddition, even though it conferred protection against the lethal effect of hyperthermia (50°C). In the pso3-1 mutant, HS induced an increase of the survival, and a decrease of the mutation frequency, after 8-MOP photoaddition and it also protected against the lethal effect of hyperthermia. The responses induced by HS were specific for 8-MOP photoaddition, since they were not observed after 254 nm ultraviolet-light damage. These results indicate that the protection conferred by HS depends of the type of lesion, and operates through the induction of different repair processes. In the pso3-1 mutant, HS could channel the repair intermediates to and error-free repair pathway.
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  • 97
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Dynamin ; Mitochondria ; GTP binding protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The isolation and characterization of MGM1, and yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.
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  • 98
    ISSN: 1432-0983
    Keywords: AEP1 ; Yeast ; Mitochindria ; ATP synthase ; PET gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36°C. The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript. In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature. A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized. This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9. The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence. Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb. None of the identified reading frames share significant homologies with entries of several data bases.
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  • 99
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    Current genetics 24 (1993), S. 179-180 
    ISSN: 1432-0983
    Keywords: Mapping ; Yeast ; Schizosaccharomyces pombe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The genetic map of the fission yeast Schizosaccharomyces pombe has been revised in the distal region of chromosome arm IIR. The spo4 locus, hitherto considered the outermost marker, has been moved to an intermediate position. As a result, and in accordance with recent physical mapping data, the order of the entire distal subgroup of some 12 genetic markers is reversed relative to previously published gene maps.
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  • 100
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    Current genetics 26 (1994), S. 281-284 
    ISSN: 1432-0983
    Keywords: Ofloxacin ; Mitochondria ; Mutation ; Recombination ; Topoisomerase ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Ofloxacin, a specific inhibitor of bacterial topoisomerase II, is known to inhibit the growth of yeast cells and to induce rho − mutants in the yeast S. cerevisiae. The frequency of ofloxacin-induced petite mutants under non-growth conditions was found to be strongly diminished when the cells were depleted in intramitochondrial ATP. Under optimal conditions of mitochondrial mutagenesis the drug induced mitotic recombination and reverse mutation in diploid strains but failed to cure either killer plasmids or the 2 μm DNA of dividing cells. The sensitivity to ofloxacin of the strains deficient in the DNA strandbreak repair pathway (rad52) was significantly higher then that of the wild-type strains and of the mutants deficient in excision or mutagenic DNA repair. The results are compatible with the idea that the cytotoxic and genetic activity of ofloxacin in yeast probably results from the inhibited DNA ligation function of topoisomerase II creating DNA breaks that are reparable through the recombination repair pathway.
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